Transdifferentiated cell populations and methods of use thereof

Abstract

Disclosed herein are methods for manufacturing transdifferentiated populations of non-pancreatic human insulin producing cells, and methods for enriching populations of non-pancreatic β-cells for cells comprising an enriched capacity for transcription factor-induced transdifferentiation into a pancreatic β-cell phenotype and function.

What is claimed is: 
     
       1. A method of manufacturing a population of transdifferentiated human insulin producing cells, the method comprising the steps of:
 (a) obtaining a population of primary liver cells; 
 (b) propagating and expanding the primary cells of step (a); 
 (c) pre-incubating the expanded cells of step (b) with a Wnt-pathway agonist, 
 (d) transdifferentiating said pre-incubated cells of step (c) by a method comprising:
 (i) contacting said pre-incubated cells with a PDX-1 polypeptide or a nucleic acid encoding a PDX-1 polypeptide, and a NeuroD1 polypeptide or nucleic acid encoding a NeuroD1 polypeptide; and 
 (ii) contacting the cells of step (i) with a MafA polypeptide or a nucleic acid encoding a MafA polypeptide; and 
 
 (e) harvesting said transdifferentiated cells; 
 wherein said method further comprises a step of incubating the cells with an epigenetic modifier, either prior to step (c), or prior, concurrent, or after step (d); 
 thereby manufacturing a population of transdifferentiated human insulin producing cells. 
 
     
     
       2. The method of  claim 1 , further comprising incubating the cells with a thyroid hormone, a TGFβ/Activin inhibitor, or a reagent that converts α-pancreatic cells to β-pancreatic cells, or any combination thereof. 
     
     
       3. The method of  claim 2 , wherein said thyroid hormone comprises T3, said TGFβ/activin inhibitor comprises Alk5i II, and said reagent that converts α-pancreatic cells to β-pancreatic cells comprises GABA. 
     
     
       4. The method of  claim 1 , wherein said epigenetic modifier comprises a histone deacetylase inhibitor (HDACi), an inhibitor of DNA methylation, a TGF0 inhibitor, or any combination thereof. 
     
     
       5. The method of  claim 4 , wherein said HDACi comprises suberanilohydroxamic acid (SAHA), sodium butyrate, romidepsin, chidamide, panobinostat, or belinostat, or any combination thereof; and wherein said TGFβ inhibitor comprises SB431542, and said inhibitor of DNA methylation comprises 5-Aza-2-deoxycitidine (5-AZA). 
     
     
       6. The method of  claim 4 , wherein said epigenetic modifier comprises a histone deacetylase inhibitor (HDACi), an inhibitor of DNA methylation, and a TGFβ inhibitor, wherein said Wnt-pathway agonist comprises a Rho kinase inhibitor, and wherein said epigenetic modifier and said Wnt-pathway agonist are added prior to step (d). 
     
     
       7. The method of  claim 1 , wherein said Wnt-pathway agonist comprises lithium (Li), Wnt9, Wnt3A, a GSK3b antagonist, a Rho Kinase inhibitor, Y27632 (Y2), or any combination thereof. 
     
     
       8. The method of  claim 1 , wherein said Wnt-pathway agonist pre-incubation occurs 24, 48, or 72 hours prior to step (d), wherein said addition of the epigenetic modifier occurs 24, 48, or 72 hours prior to the transdifferentiation step (d), or wherein said addition of the epigenetic modifier and said pre-incubation with Wnt-pathway agonist are concurrent, or any combination thereof. 
     
     
       9. The method of  claim 1 , wherein said method further comprises
 contacting said pre-incubated cells with an additional pancreatic transcription factor at step (i); or 
 contacting said pre-incubated cells with an additional pancreatic transcription factor at step (ii); or 
 contacting said pre-incubated cells with an additional pancreatic transcription factor at both step (i) and step (ii). 
 
     
     
       10. The method of  claim 9 , wherein said additional pancreatic transcription factor comprises PAX4, PAX6, ILS-1, NGN3, NKX6.1, RFX6, or FOXA2. 
     
     
       11. The method of  claim 1 , further comprising incubating the cells with nicotine amide, epidermal growth factor (EGF), exendin-4, or any combination thereof, during step (d). 
     
     
       12. A method for enriching a population of primary liver cells comprising enriched capacity for transcription factor-induced transdifferentiation into a pancreatic β-cell like phenotype and function, said method comprising the steps of:
 (a) obtaining a population of primary human liver cells; 
 (b) identifying cells within the population of (a) having increased expression of at least one gene of the group comprising a solute carrier family 2, facilitated glucose transporter member 3 (GLUT-3); a vesicle-associated membrane protein 2 (VAMP2); a syntaxin-1A (Stx1a); a tyrosine-protein kinase transmembrane receptor ROR2 (ROR2); Frizzled-4 (FZD4); a pituitary homeobox 2 (PITX2); and 
 a Proto-oncogene Wnt-1 (WNT1); or any combination thereof, wherein said increased expression is compared within the non-pancreatic β-cell population; and 
 (c) selecting said liver cells having increased expression of at least one gene recited in (b); 
 wherein said selected cells comprise an enriched capacity for transcription factor induced transdifferentiation to a pancreatic β-cell like phenotype and function. 
 
     
     
       13. The method of  claim 12 , wherein said identifying further comprises identifying cells with increased expression of at least one gene selected from the group comprising vesicle-associated membrane protein 4 (VAMP4); thrombospondin-1; discoidin, CUB and LCCL domain-containing protein 2 (THBS1); integrin alpha-6 (ITGA6); homer protein homolog 1 (HOMER1); lysosome-associated membrane glycoprotein 3 (LAMP3); bone morphogenetic protein receptor type-2(BMPR2); or with decreased expression of at least one gene selected from the group comprising multidrug resistance protein 1 (ABCB1), integrin alpha-4 (ITGA4), and phosphatidylcholine translocator ABCB4 ABCB4), or any combination thereof. 
     
     
       14. The method of  claim 12 , said method further comprising steps of
 (a) propagating and expanding said enriched primary liver cells; 
 (b) pre-incubating the expanded cells of step (a) with a Wnt-pathway agonist; 
 (c) incubating said pre-incubated cells with at least one epigenetic modifier; and 
 (d) collecting said cells. 
 
     
     
       15. The method of  claim 14 , wherein said epigenetic modifier comprises a histone deacetylase inhibitor (HDACi), an inhibitor of DNA methylation, a TGFβ inhibitor, suberanilohydroxamic acid (SAHA), sodium butyrate, romidepsin, chidamide, panobinostat, belinostat, SB431542, 5-Aza-2-deoxycitidine (5-AZA), or said Wnt-pathway agonist comprises lithium (Li), Wnt9, Wnt3, a GSK3b antagonist, a Rho Kinase inhibitor comprises Y27632 (Y2), or any combination thereof. 
     
     
       16. The method of  claim 14 , wherein said incubating with said epigenetic modifier occurs before said pre-incubation, at the same time as said pre-incubation, following said pre-incubation and at the same time than a transdifferentiation step, or following a transdifferentiation step. 
     
     
       17. The method of  claim 14 , wherein said Wnt-pathway agonist pre-incubation occurs 24, 48, or 72 hours prior to a transdifferentiation step. 
     
     
       18. The method of  claim 14 , wherein said identifying comprises incubating cells with a labeled antibody or ligand that binds the protein product of said at least one gene and wherein said selecting comprises selecting cells bound to said labeled antibody or ligand. 
     
     
       19. The method of  claim 12 , wherein following transcription factor induced transdifferentiation of said selected cells, said transdifferentiated selected cell population expresses increased endogenous Nkx6.1, increased insulin content, increased glucagon content, or increased glucose-regulated insulin secretion and C-peptide secretion, or any combination thereof, compared with a control transdifferentiated non-selected population of cells.